A Novel Transmembrane Serine Protease (TMPRSS3) Overexpressed in Pancreatic Cancer1,2

[CANCER RESEARCH 60, 2602–2606, May 15, 2000] Advances in Brief

Christine Wallrapp, Susanne Ha¨hnel, Friederike Mu¨ller-Pillasch, Beata Burghardt, Takeshi Iwamura, Manuel Ruthenbu¨rger, Markus M. Lerch, Guido Adler, and Thomas M. Gress3 Department of Internal Medicine I, University of Ulm, 89081 Ulm, Germany [C. W., S. H., F. M-P., G. A., T. M. G.]; Hungarian Academy of Sciences, University of Budapest, 1450 Budapest, Hungary [B. B.]; Department of Medicine B, University of Mu¨nster, 48129 Mu¨nster, Germany [M. M. L., M. R.]; Miyazaki Medical College, Kiyotake, Miyazaki 889-1692, Japan [T. I.]

Abstract expressed in pancreatic cancer compared with normal pancreas and chronic pancreatitis tissue. From the 16 gene fragments isolated in this We report the characterization of a novel serine protease of the chy- study, we selected the 313-bp gene fragment RDA12 (GenBank motrypsin family, recently isolated by cDNA-representational difference accession no. U54603) for further characterization. Database compar- analysis, as a gene overexpressed in pancreatic cancer. The 2.3-kb mRNA of the gene, named TMPRSS3, is strongly expressed in a subset of pan- ison revealed a moderate homology to a number of serine proteases, creatic cancer and various other cancer tissues, and its expression corre- indicating that RDA12 may be a fragment of a novel protease with lates with the metastatic potential of the clonal SUIT-2 pancreatic cancer cancer-specific expression. cell lines. The deduced polypeptide sequence consists of 437 amino acids and exhibits all of the structural features characteristic of serine proteases Materials and Methods with trypsin-like activity. TMPRSS3 is membrane bound with a NH - 2 Materials. Human tissue from patients with ductal adenocarcinoma of the terminal signal-anchor sequence and a glycosylated extracellular region pancreas (n ϭ 13), carcinoma tissues of different origin, human pancreatic containing the serine protease domain. Thus, TMPRSS3 is a novel mem- tissue from organ donors (n ϭ 6), and chronic pancreatitis tissue (n ϭ 6) was brane-bound serine protease overexpressed in cancer, which may be of provided by the Hungarian Academy of Sciences (Budapest, Hungary) and the importance for processes involved in metastasis formation and tumor Department of Surgery of the University of Ulm. All tissue samples were invasion. obtained after approval by the local Ethics Committee. The human pancreatic cancer cell lines were obtained from the following Introduction suppliers: PATU-8988S and PATU-8988T (German Collection of Microor- Proteases have been increasingly recognized as important factors in ganisms and Cell Cultures, Braunschweig, Germany); PANC-1 and MIA- the pathophysiology of tumorous diseases. The proteolytic degrada- PaCa-2 (European Collection of Animal Cell Cultures, Salisbury, United Kingdom); HPAF (Metzgar, Durham, NC); Capan-1, Capan-2, and AsPC-1 tion of the extracellular matrix, which is an indispensable step in (Cell Lines Service, Heidelberg, Germany); Patu II (Elsa¨sser, Marburg, Ger- tumor invasion and metastasis, is mediated by members of the four many); PC2 (Bu¨low, Mainz, Germany); SUIT-2 (S2-007, S2-013, S2-020, and major classes of endopeptidases, including serine, cysteine, aspartyl, S2-028; Iwamura, Miyazaki, Japan; Ref. 4); and SKPC2 and IMIM-PC2 and metalloproteases (1). In this highly complicated process, a cas- (P. Real, IMIM, Barcelona, Spain). cade of events requiring a variety of proteases seems to be involved. Cloning of a New Serine Protease cDNA. In a recent screen for differ- Numerous reports have demonstrated an increased production of entially expressed genes in pancreatic carcinoma, the 313-bp gene fragment extracellular matrix degrading enzymes, including type IV collagen- RDA12 (accession no. U54603) was isolated by cDNA-RDA (3); this fragment ase (MMP-2), cathepsin B, cathepsin D, and serine proteases such as encodes the putative motif of a new serine protease. The RDA12 fragment was plasminogen activator in tumor cells (1). The proteolytic enzymes of used to screen ϳ20,000 clones of an oligo(dT)-primed cDNA library from a the serine protease family exist as single-chain or double-chain zy- pancreatic cancer cell line by hybridization. Both strands of the longest cDNA clone, RDA12/2, were sequenced by primer walking. For stable transfection in mogens activated by specific and limited proteolytic cleavage. They mammalian cells, the cDNA clone RDA12/2 was cloned in sense and antisense contain the three active-site amino acids histidine, aspartate, and orientation into the BamHI site of the mammalian expression vector pH␤- serine, which participate in peptide bond hydrolysis. The geometric Apr1-neo (5). A COOH-terminal-tagged TMPRSS3 expression vector was orientation of this catalytic triad is similar in different serine constructed by insertion of a 1427-bp fragment (nucleotides 96–1522) con- proteases, despite the fact that folding of the proteases may be taining the open reading frame of TMPRSS3 into the BstXI site of the mammalian different (2). expression vector pcDNA6/V5/His B (Invitrogen, San Diego, CA). In the present study, we report the cloning and characterization of Northern Blot Analyses. The expression of TMPRSS3 was studied by a novel serine protease identified in a recent cDNA-RDA4 approach hybridizations using Northern blots containing 30 ␮g each of total RNA from (3). This study was designed to isolate gene fragments highly over- normal pancreas tissue, chronic pancreatitis tissue, different carcinoma tissues, and cell lines. The Northern blots containing RNA of different human tissues were purchased from Clontech (Heidelberg, Germany). Received 12/17/99; accepted 3/30/00. Cell Culture and Transfection. For functional analysis of TMPRSS3, the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with S2-020 pancreatic cancer cell line, which expresses no endogenous TMPRSS3 18 U.S.C. Section 1734 solely to indicate this fact. mRNA, was transfected with the TMPRSS3-pH␤-Apr1-neo construct in sense 1 This work was supported by grants from the Bundesministerium fu¨r Bildung und and antisense orientation using DMRIE-C (Life Technologies, Inc., Eggen- Forschung (01 GB9401), the European Community (BMH4-CT98-3085), and the Deut- stein, Germany). Several clones were picked that showed various degrees of sche Forschungsgemeinschaft (SFB518, project B1; to T. M. G.). 2 The nucleotide sequence in this report has been submitted to the GenBank Data stable TMPRSS3 sense/antisense mRNA expression. Two of each sense and Library with accession no. AF179224. antisense clones were used for functional assays. 3 To whom requests for reprints should be addressed, at Department of Internal HEK-293 cells were plated at 1.5 ϫ 106 cells/10-cm dish and grown Medicine I, University of Ulm, 89081 Ulm, Germany. Phone: 49-731-5024385; Fax: overnight in DMEM supplemented with 10% FCS. Cells were transiently 49-731-5024302; E-mail: [email protected]. 4 The abbreviations used are: RDA, representational difference analysis; PNGase F, transfected with the TMPRSS3-pcDNA6/V5/His plasmid DNA by use of the peptide-N-glycosidase F. calcium phosphate protocol. 2602

Fig. 1. Nucleotide sequence of the cDNA coding for human TMPRSS3 and its predicted amino acid sequence. The bold nucleotide sequence 1189–1501 represents the initially isolated RDA12 gene fragment, the underlined nucleotides 2045–2050 mark the potential polyadenylation signal. The amino acid sequence highlighted by a gray box represents the potential transmembrane domain. Œ indicates the active-site residues histidine (H), aspartate (D), and serine (S). Double underlines indicate potential N-linked glycosylation sites.

Preparation of Cell Extracts and Subcellular Fractionation. Forty-eight Glycosylation. For PNGase F treatment, immunopurified protein was in- h after transient transfection with V5-tagged TMPRSS3 into HEK-293 cells, cubated overnight with 2 units of PNGase F supplemented with 10 mM EDTA protein extracts were prepared by resuspending pelleted cells in 1% Triton at 37°C. Inhibition of N- and mucin-like O-glycosylation was performed by X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 50 mM Tris-HCl cultivating TMPRSS3-expressing HEK-293 cells for 24 h in DMEM, 10% (pH 7.2) supplemented with 5 ␮g/ml Aprotinin, 5 mM Pefabloc, and 10 ␮g/ml FCS containing either 2.5 ␮g/ml tunicamycin (7) or 2 mM phenyl-N-Acetyl- Pepstatin. For immunopurification of the epitope-tagged protein, cell lysates ␣-D-galactosaminide (8). Thereafter, cells were harvested for protein extrac- were incubated with V5 antibody conjugated to protein G-agarose beads at 4°C tion. for4honashaker. The agarose beads were pelleted by centrifugation and Functional Assays. Nude mouse experiments were done by injecting washed twice with 150 mM NaCl, 5 mM EDTA, 50 mM Tris ϩ 0.1% NP40. The 2 ϫ 106 S2-020 cells stably transfected with TMPRSS3 sense/antisense con- washed pellets were resuspended in 150 mM NaCl, 5 mM EDTA, 50 mM structs, both s.c. and in the tail vein of female nu/nu mice. Five weeks after the Tris ϩ 0.1% NP40 for PNGase F treatment. tail vein injections, the lung, spleen, and liver were used for standard histo- Subcellular fractions were prepared from transiently transfected HEK-293 logical analysis to identify the presence or absence of metastatic lesions. cells as reported previously (6). The plasma membrane-enriched fraction, Subcutaneous tumors were measured and used for histological analysis. which was prepared using sucrose density gradient centrifugation, the cytosolic In vitro matrigel invasion assays were done by seeding 105 transfected cells fraction, and concentrated culture medium were studied by Western blot in medium ϩ 1% FCS in the upper chamber of Matrigel-coated 8-␮m tran- analysis. swell plates. The lower chamber was filled with medium ϩ 10% FCS. The 2603

Fig. 2. Northern blot analyses of the TMPRSS3 transcript in different tissues and cell lines. The Northern blots contain 30 ␮g of total RNA per lane from normal human pancreas (n ϭ 6), chronic pancreatitis tissue (n ϭ 6), pancreatic carcinoma tissue (n ϭ 13; Lanes 1–13), and cancer tissues of different origin (Lanes 14–16, 19–21, and 23, colorectal carcinoma; Lanes 17 and 25–27, gastric cancer; Lane 22, soft tissue sarcoma; Lane 18, breast cancer; Lane 24, carcinoma of the papilla vateri) and the SUIT-2 subclones S2-028, S2-013, and S2-007. RNAs from normal pancreas, chronic pancreatitis, and pancreatic cancer tissue samples were run on the same Northern blot gels. The autoradiographs for cancer and control tissues are shown sepa- rately for improved presentation of the data.

number of invading cells adhering to the lower side of the porous membrane TMPRSS3 is predicted to cleave in a trypsin-like manner after was counted after fixation with 4% paraformaldehyde and staining with lysine or arginine residues because it contains D381 at the base of the methylene blue. specificity pocket that binds the substrate (13). In addition, the novel The proteolytic activity in TMPRSS3 sense/antisense-transfected S2-020 protein shares considerable structural similarities of the TMPRSS cells and transiently transfected HEK-293 cells was determined fluorometri- family, including the putative NH -terminal membrane anchor and the cally in native lysates and lysates treated with enterokinase for activation, 2 conserved cysteine residues, which by homology most likely form the using oligopeptide substrates for elastase-like (Ala-Ala-Ala-Ala) and trypsin- 196 310 230 246 356 372 383 410 like (Ile-Pro-Arg) serine proteases as described previously (9). disulfide bonds C –C ,C –C ,C –C , and C –C . Chromosomal Mapping of the TMPRSS3 Gene Locus. The chromo- Serine proteases are most commonly synthesized as inactive proen- somal localization of TMPRSS3 was determined by screening the GeneBridge4 zymes, which are activated by extracellular, proteolytic removal radiation hybrid panel (Research Genetics, Huntsville, AL), using the of a propeptide. At the NH2-terminal part of the protease domain, TMPRSS3-specific primers 5Ј-CATGTGGTGGGCATCGTTA-3Ј and 5Ј- TMPRSS3 contains the peptide sequence RVVGG, which is typical CCAGTTGAGATAGGCTGAG-3Ј. for the proteolytic activator site of many protease zymogens. The potential cleavage between R204 and V205 would result in a new Results and Discussion terminal ␣-amino group, which forms a salt bridge with D386 and The 313-bp fragment encoding the putative motif of a new serine thereby leads to the assembly of the functional catalytic sites. There- protease isolated in a recent cDNA-RDA screen for genes differen- fore, the activated form would consist of a non-protease and a protease tially expressed in pancreatic cancer (3) was used to screen a pancre- subunit linked by a disulfide bond that most likely involves C196– atic cancer cDNA library. Among 16 isolated homologous clones, a C310. Whether this activation is mediated under physiological condi- clone designated RDA12/2 contained the full-length sequence. The tions by autocatalytic cleavage or other proteases is not known. The sequence of clone RDA12/2 comprised 2071 bp, including a 214-bp TMPRSS3 gene locus was localized to chromosome 11 at q23.3 5Ј untranslated region, an open reading frame of 1311 nucleotides, between the markers D11S4362 and D11S4387 by use of a radiation and a 546-bp 3Ј untranslated region (Fig. 1). Translation of the open hybrid panel. reading frame suggests that the cDNA codes for a putative polypep- As anticipated, an overexpression of the 2.3-kb transcript was tide of 437 amino acids with an estimated molecular mass of 48.202 found in 9 of 13 primary pancreatic carcinoma tissues (Fig. 2) and in 10 of 16 pancreatic carcinoma cell lines (not shown) by Northern blot kDa. The NH2-terminal region of the hypothetical protein contains a putative signal-anchor sequence characteristic for group II integral analysis. Because TMPRSS3 was not expressed in normal pancreas membrane proteins. The highly hydrophobic region of 22 amino acids (n ϭ 6) and in chronic pancreatitis (n ϭ 6) tissue samples, overex- may serve as a transmembrane domain that is involved in anchoring pression appears to be cancer-specific and not due to inflammatory the protease to the cell membrane. According to the charge difference alterations in the stroma. No clear correlation was found between the rule (10), it can be assumed that the COOH terminus of the protein stage of pancreatic tumors and the expression of the protease (Table with its protease module is located on the extracellular surface. 1). Northern blot analyses with RNA from a small number of other Although the nucleotide sequence is unique, database comparisons tumor tissues revealed that TMPRSS3 overexpression is not restricted of the amino acid sequence revealed a homology to a number of serine proteases. Thirty-five percent identity and ϳ50% similarity was found to members of the serine protease family known as the human trans- Table 1 TNM classification of pancreatic cancer patients membrane proteases, TMPRSS1/hepsin (11) or TMPRSS2 (12). Thus, Tissue sample TNM classification our new protease is the third member of a family of transmembrane- 1T3N1M0 bound serine proteases. Consequently, this new gene was named 2T3N1M0 3TN M TMPRSS3 for transmembrane protease, serine 3. Sequence homology 2 1 0 4T2N0Mx was high in the domains containing the three principal active-site 5T3N1Mx amino acids H245,D290, and S387, required for peptide bond hydrol- 6T2N1M0 7T2N1M0 ysis. The arrangement of the catalytic residues in the linear sequence 8T2N0M0 defines the membership of TMPRSS3 to the S1 family of the chy- 9T3N1M0 10 T N M motrypsin clan SA of serine-type peptidases (2). The prototype of this 3 1 0 11 T3N1M0 family is chymotrypsin, and the three-dimensional structures of some 12 T4N0M1 of its members have already been resolved (12). 13 T2N1M1 2604

Fig. 3. a, hydropathicity plot of the predicted TM- PRSS3 protein. The method of Kyte and Doolittle (20) was used, using a window of 17 residues (http:// bioinformatics.weizmann.ac.il/hydroph/). The peak spanning amino acids 32–53 represents the putative transmembrane domain. b, schematic representation of the different domains of TMPRSS3, a type II membrane-associated serine protease. Numbers cor- respond to the amino acids, deduced from the cDNA sequence shown in Fig. 1. The disulfide bonds were deduced based on the structure of TMPRSS1 and TMPRSS2, the most homologous proteins. pot., potential.

to pancreatic cancer, but can also be found in gastric (n ϭ 4), expressed in HEK-293 cells, immunoprecipitated, and treated with colorectal (n ϭ 7), and ampullary (n ϭ 1) cancer. No expression was PNGase F. This resulted in an increase in mobility on denaturing found in one tissue sample each of soft tissue sarcoma and breast SDS-PAGE, demonstrating N-glycosylation of TMPRSS3 (Fig. 4). cancer (Fig. 2). TMPRSS3 transcripts were not detectable in normal Cultivation of transfected HEK-293 cells in the presence of tunica- heart, brain, placenta, lung, liver, skeletal muscle, uterus, and adipose mycin, an inhibitor of N-glycosylation, revealed the same mobility tissue. A weak signal was found in tissues of the normal gastrointes- shift of TMPRSS3 to a molecular mass of 60 kDa. Phenyl-N-acetyl- tinal tract (esophagus, stomach, small intestine, colon) and in some ␣-D-galactosaminide, which inhibits mucin-like O-glycosylation, had tissues of the urogenital tract (kidney and bladder). Nevertheless, no effect on the molecular mass (data not shown). The generation of expression was much weaker than in the corresponding tumors (data recombinant proteases frequently has been shown to be difficult or not shown). Furthermore, we analyzed the expression of TMPRSS3 in impossible (14). Despite extensive and repeated efforts, we were the SUIT-2 clonal cell lines S2-007, S2-013, and S2-028 (4). These unable to successfully generate recombinant protein in Escherichia subclones of the human pancreatic cancer cell line SUIT-2 differ in coli and insect cells, possibly because TMPRSS3, as many other their spontaneous metastatic potential after s.c. injection in nude mice. proteases, had a cytotoxic effect on transfected cells. Repeated efforts In this setting S2-007 regularly shows a high rate of metastases, to generate peptide antisera failed as well (data not shown), and a whereas the other two cell lines show a lower rate (S2-013) or no TMPRSS3 antibody was therefore not available for further studies. metastases at all (S2-028). As shown in Fig. 2, the strength of Whereas the established physiological role of the chymotrypsin TMPRSS3 expression correlated well to the metastatic potential of the family of secreted serine proteases is primarily in protein catabolism, SUIT-2 subclones, which may serve as an indication that this serine the function of serine proteases of the TMPRSS family is of special protease is associated with the promotion of metastasis. interest. Although the function of TMPRSS2 remains unknown (12, The sequence of TMPRSS3 suggests that this novel serine protease 15), TMPRSS1, also known as hepsin, frequently is overexpressed in contains a signal anchor characteristic for group II integral membrane proteins with a hydrophobic transmembrane domain (Fig. 3a). Ac- cording to the charge difference rule (10), the transmembrane domain (amino acids 32–53) anchors the protease to the cell membrane.

Because of this anchorage, the NH2-terminal domain (amino acids 1–31) would appear to be located intracellularly, and the COOH- terminal region (amino acids 54–437), which contains the catalytic domain, would be located extracellularly (Fig. 3b). The alleged subcellular localization of the protease was confirmed using a V5-tagged TMPRSS3 construct, which was transiently transfected into HEK-293 cells. Membrane fractionation and Western blotting with the corresponding anti-V5 antibody revealed a signal only in the plasma membrane-enriched fraction, whereas no tagged TMPRSS3 protein was detectable in the cytosol and in the culture medium (Fig. 4). This experiment also uncovered post-translational modifications of Fig. 4. Western blot analysis of V5-tagged TMPRSS3 protein. Protein extracts from TMPRSS3. Although the calculated theoretical molecular mass of the TMPRSS3-pcDNA6/V5/His-transfected HEK-293 cells were resolved in 9% SDS-PAGE epitope-tagged fusion protein is 52 kDa, its size in a SDS-polyacryl- and transferred to nitrocellulose membranes. Membranes were immunoblotted with an anti-V5-horseradish peroxidase antibody followed by chemiluminescence detection. a,20 amide gel is ϳ68 kDa, suggesting the presence of potential carbohy- ␮g of total protein extract. b, subcellular localization; C, cytosolic fraction; M, plasma drate moieties. The primary sequence of TMPRSS3 displays two membrane-enriched fraction; S, concentrated culture medium. c, analysis of N-linked 130 glycosylation of the TMPRSS3 protein. A shift in molecular mass was detected both after consensus motifs for N-linked glycosylation (N-X-T/S) at N and PNGase F treatment of the immunoprecipitated protein and after exposure of the trans- N178. To confirm this N-glycosylation, epitope-tagged TMPRSS3 was fected cells to tunicamycin, indicating N-glycosylation of the protein. 2605

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2000 American Association for Cancer Research. NOVEL SERINE PROTEASE IN PANCREATIC CANCER ovarian tumors and may therefore contribute to the invasive nature or this novel serine protease are unknown and the specific substrates growth capacity of ovarian tumor cells (16). Treatment of hepatoma have not yet been identified, the precise role of TMPRSS3 in carci- cells with antihepsin antibodies or specific antisense oligonucleotides nogenesis remains to be elucidated. confirmed that hepsin plays an essential role in cell growth and Acknowledgments maintenance of cell morphology (17). It has also been shown that hepsin can proteolytically activate human coagulation factor VII and We thank G. Adler for continual support, U. Lacher for excellent technical thereby contribute to the activation of the coagulation cascade (18). assistance, M. A. Hollingsworth for the pH␤-Apr1-neo vector, and F. Gan- The correlation of TMPRSS3 expression with the metastatic poten- sauge and G. Varga for providing human pancreatic tissue samples. tial of the SUIT-2 cell lines is a first indication that this new protease, References in the same way as hepsin, may be involved in promoting metastasis formation and tumor invasion. To confirm this hypothesis in func- 1. DeClerck, Y. A., and Imren, S. Protease inhibitors: role and potential therapeutic use in human cancer. Eur. J. Cancer, 30A: 2170–2180, 1994. tional assays, stably transfected S2-020 cell lines were generated 2. Rawlings, N. D., and Barrett, A. J. Families of serine peptidases. Methods Enzymol., using the TMPRSS3 cDNA cloned in sense and antisense orientation 244: 19–61, 1994. into the pH␤-Apr1-neo vector. Several clones were generated show- 3. Gress, T. M., Wallrapp, C., Frohme, M., Muller-Pillasch, F., Lacher, U., Friess, H., Buchler, M., Adler, G., and Hoheisel, J. D. 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Biochemistry, 27: 1067–1074, 1988. fected cells because we lacked a specific antibody. In the absence of 12. Paoloni Giacobino, A., Chen, H., Peitsch, M. C., Rossier, C., and Antonarakis, S. E. Cloning of the TMPRSS2 gene, which encodes a novel serine protease with trans- final experimental proof, we can therefore only hypothesize, based on membrane, LDLRA, and SRCR domains and maps to 21q22.3. Genomics, 44: the structural characteristics and the expression pattern in cancer 309–320, 1997. 13. Steitz, T. A., Henderson, R., and Blow, D. M. Structure of crystalline ␣-chymotryp- tissues and in the SUIT-2 subclones, that this new protease has a sin. 3. Crystallographic studies of substrates and inhibitors bound to the active site of potential role for tumor progression, metastasis formation, and tumor ␣-chymotrypsin. J. Mol. Biol., 46: 337–348, 1969. invasion. 14. Anisowicz, A., Sotiropoulou, G., Stenman, G., Mok, S. C., and Sager, R. 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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2000 American Association for Cancer Research. A Novel Transmembrane Serine Protease (TMPRSS3) Overexpressed in Pancreatic Cancer 1,2

Christine Wallrapp, Susanne Hähnel, Friederike Müller-Pillasch, et al.

Cancer Res 2000;60:2602-2606.

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